Project Summary/Abstract Little is known about the transcriptional regulatory mechanisms directing the differentiation of presecretory ameloblasts (PAB) into secretory ameloblasts (SAB) and amelogenin secretion. SABs are responsible for the synthesis and secretion of enamel matrix proteins (EMPs) (primarily amelogenins) to form the full thickness of enamel matrix. For secretion, EMPs are packaged into secretory vesicles, transported to Tomes' process, the characteristic cytoplasmic projections of SAB, then exocytosed into the enamel space to direct hydroxyapatite crystal growth. We have found that special AT-rich sequence binding protein 1 (SATB1), a genome organizer which regulates chromatin architecture and gene expression, is highly expressed in PAB, and that in the absence of SATB1, differentiation of PAB to the polarized secretory SAB is inhibited. SABs in Satb1-/- mice lack cell polarity, apical actin filament assembly, and Tomes' process formation. These Satb1-/- SABs display major defects in amelogenin secretion, resulting in thin and hypomineralized enamel. These findings imply that SATB1 may govern enamel formation by regulating the expression of genes required for amelogenin trafficking. Our preliminary studies identified synaptoporin (SYNPR) and epidermal growth factor receptor pathway substrate 8 (EPS8) as SATB1-dependent genes that potentially mediate amelogenin secretion and trafficking. EPS8, required for actin filament assembly in the formation of intestinal microvilli, was immunolocalized in Tomes' processes, and SYNPR, a vesicle component, was found associated with the vesicle-like structures in the cytoplasm of SAB, where the most of vesicles carry amelogenins. Thus, we hypothesize that SATB1 regulates amelogenin secretory trafficking through its target genes associated with the formation of secretory vesicles (via SYNPR) and the actin filament assembly/Tomes' process formation (via EPS8). To address this hypothesis we propose two specific aims: Specific Aim 1: We will determine whether synaptoporin is necessary for amelogenin-containing vesicle formation in ameloblasts. By immunostaining and confocal microscope imaging of wt, Satb1-/- and Synaptoporin-/- mouse SAB, we will determine if synaptoporin- containing vesicles carry amelogenins. In parallel, we will perform SAB vesicle composition analysis to verify the results. Specific Aim 2: We will investigate the roles of SATB1 and EPS8 on apical actin filament assembly in Tomes' process formation and amelogenin secretion. By knockdown and inducible overexpression strategies using the human PAB primary cell culture on 3-D, mechanisms by which SATB1 regulates EPS8 to direct ameloblast morphogenesis and protein secretion will be investigated. How SATB1 regulates chromatin and epigenetic statuses of the loci of Eps8 and other target genes will also be studied. These studies will advance our understanding how ameloblasts differentiate to form enamel, and how SATB1 regulates gene expression and chromatin structure for key players in these processes. The information from this research may provide an insight to help engineer secretory ameloblasts to regenerate enamel tissues.